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. 2016 Mar;75(3):617-22.
doi: 10.1136/annrheumdis-2014-206297. Epub 2015 Feb 5.

Interaction between innate immunity and Ro52-induced antibody causes Sjögren's syndrome-like disorder in mice

Barbara M Szczerba  1 , Paulina Kaplonek  2 , Nina Wolska  2 , Anna Podsiadlowska  2 , Paulina D Rybakowska  3 , Paromita Dey  4 , Astrid Rasmussen  2 , Kiely Grundahl  2 , Kimberly S Hefner  5 , Donald U Stone  6 , Stephen Young  7 , David M Lewis  8 , Lida Radfar  8 , R Hal Scofield  9 , Kathy L Sivils  2 , Harini Bagavant  3 , Umesh S Deshmukh  3
Affiliations

Interaction between innate immunity and Ro52-induced antibody causes Sjögren's syndrome-like disorder in mice

Barbara M Szczerba et al. Ann Rheum Dis. 2016 Mar.

Abstract

Objectives: Autoantibodies reactive with Ro52 are often found in sera of patients with Sjögren's syndrome (SS). This study was undertaken to investigate the role of Ro52-induced immune responses in pathogenesis of SS.

Methods: New Zealand Mixed (NZM) 2758 mice were immunised with Ro52 in alum adjuvant. Control mice were immunised either with maltose-binding protein or injected with alum alone. Mice were monitored for anti-Ro52 antibody, sialoadenitis and pilocarpine-induced salivation. Antibody binding to salivary gland (SG) cells was analysed in vivo and in vitro by immunofluorescence. Sera from immunised mice were passively transferred into untreated or alum injected NZM2758 mice.

Results: By day 30 post-immunisation, Ro52 immunised mice generated immunoprecipitating anti-Ro52 antibodies and they had the maximum drop in saliva production. Both Ro52 immunised and control mice showed evidence of mild sialoadenitis. However, only Ro52 immunised mice had antibody deposition in their SG. Passive transfer of Ro52-immune sera induced SG dysfunction in recipient mice, only if the recipients were primed with alum. In vitro, antibodies from Ro52-immune sera were internalised by a SG cell line and this uptake was inhibited by cytochalasin D treatment.

Conclusions: Our data show for the first time that antibodies induced by Ro52 are capable of inducing SG dysfunction, and that this phenomenon is dependent on the activation of innate immunity. The mouse model described in this study implies that autoantibody deposition in the SG might be an important step in the induction of xerostomia and pathogenesis of SS.

Keywords: Autoantibodies; Autoimmunity; Sjögren's Syndrome.

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Figures

Figure 1
Figure 1. SG dysfunction is most prominent in Ro52 immunized mice and it correlates with levels of anti-Ro52 antibody
A. Immunoprecipitating anti-mRo52 antibodies are generated in 7 out of 8 NZM2758 mice immunized with Ro52. Data is represented as ΔCPM, which is CPM with serum sample minus CPM without serum. None of the MBP immunized mice have antibodies over the cutoff, which is mean ΔCPM+2SD for MBP immunized mice. Mann-Whitney test was used to determine the statistical significance. B. In comparison with untreated mice, Ro52 immunized mice show a 65% drop and MBP immunized mice show a 28% drop in mean saliva volumes. Kruskal–Wallis test was used to determine the statistical significance and ****indicates p<0.0001. The number of mice used provides the experiment >99% power at a significance level (alpha) of 0.05 (two-tailed) C. Inverse correlation is seen between saliva volume and level of anti-Ro52 antibody (p=0.0016; r = −0.802) as determined by Spearman correlation test.
Figure 2
Figure 2. Mild sialoadenitis was seen in NZM2758 mice injected either with Ro52 or MBP, but IgG deposition was only seen in Ro52 immunized mice
A, B. H&E stained sections of SMG obtained on day 35 from MBP immunized (A) and Ro52 immunized mouse (B) showing mild sialoadenitis. C, D. Representative pictures of direct immunofluorescence staining of SMG from mice injected either with MBP (C) or with Ro52 (D). IgG deposition was detected by direct immunofluorescence using FITC conjugated goat anti-mouse IgG. Scale bar = 50μm.
Figure 3
Figure 3. Passive transfer of immune sera from Ro52 immunized mice rapidly induces SG dysfunction only in recipients primed with alum
NZM2758 mice were either untreated (left panel) or injected with alum (right panel). On day 30, sera obtained from mice immunized either with alum alone, or MBP or Ro52 were used for passive transfer and saliva production was measured after 24h. There was no drop in saliva production in untreated mice receiving Ro52-immune sera. Compared to untreated mice, all groups of alum injected mice showed a drop in mean saliva volume. Of the mice pre-injected with alum, only transfer of Ro52-immune sera led to a significant reduction in saliva production. (p = 0.0075, Kruskal–Wallis test).
Figure 4
Figure 4. Antibodies from Ro52-immune sera are actively internalized by SG cell line
SCA9-15 cells were grown on coverslips and incubated with immune sera at 1:250 dilution. Bound antibodies were detected with FITC conjugated goat anti-mouse IgG (A & D, green) and nuclei were stained with DAPI (B & E, blue). Antibodies from Ro52-immune sera show strong cytoplasmic staining in comparison with MBP-immune sera (C & F, overlay). Scale bar = 20μm.
Figure 5
Figure 5. Cytochalasin D treatment prevents internalization of antibodies by SCA9-15 cells
Confocal images show that cells incubated with Ro52-immune sera have cytoplasmic IgG (green) staining (A), which is not seen in cells incubated with control sera (C). Cytochalasin D treatment localizes antibody reactivity to the surface (B). Cells treated with Cytochalasin D, become rounded due to collapse in actin filaments (red) stained with Phalloidin Alexa Fluor 568. Similar results were obtained in 3 additional experiments. Scale bar = 10μm.
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References

    1. Reksten TR, Jonsson MV. Sjögren’s syndrome: an update on epidemiology and current insights on pathophysiology. Oral Maxillofac Surg Clin North Am. 2014;26:1–12. - PubMed
    1. Kyriakidis NC, Kapsogeorgou EK, Tzioufas AG. A comprehensive review of autoantibodies in primary Sjögren’s syndrome: Clinical phenotypes and regulatory mechanisms. J Autoimmunity. 2014;51:67–74. - PubMed
    1. Ozato K, Shin DM, Chang TH, et al. TRIM family proteins and their emerging roles in innate immunity. Nat Rev Immunol. 2008;8:849–60. - PMC - PubMed
    1. Kong HJ, Anderson DE, Lee CH, Jang, et al. Cutting edge: autoantigen Ro52 is an interferon inducible E3 ligase that ubiquitinates IRF-8 and enhances cytokine expression in macrophages. J Immunol. 2007;179:26–30. - PubMed
    1. Higgs R, NíGabhann J, Ben Larbi N, et al. The E3 ubiquitin ligase Ro52 negatively regulates IFN-beta production post-pathogen recognition by polyubiquitin-mediated degradation of IRF3. J Immunol. 2008;181:1780–6. - PMC - PubMed

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